POS0486 IDENTIFICATION OF NEW CANDIDATE TARGETS INVOLVED IN ACTIVATION OF CARDIAC FIBROBLASTS UNDER IMMUNOFIBROTIC CONDITIONS
Authors: I. Kocherova, E. Pachera, D. Nurzynska, F. DI Meglio, O. Distler, P. Blyszczuk, G. Kania
Journal: Annals of the Rheumatic Diseases (2022)

Background Inflammatory dilated cardiomyopathy (iDCM) often leads to heart failure (HF), which is the main cause of mortality in patients with systemic diseases. Fibroblast activation, driven by the activator protein 1 family member Fos-related antigen 2 (FOSL2), represents a critical step in cardiac fibrogenesis. The existing antifibrotic therapies, based on known fibrotic markers, failed to demonstrate efficacy against myocardial fibrosis. Objectives To identify new candidate targets implicated in cardiac fibrogenesis under immunofibrotic conditions. Methods Cardiac fibroblasts were isolated from the left atria of patients (n=5) undergoing heart transplantation due to HF associated with iDCM and from unaffected hearts of brain-dead donors (UDs, n=5). Protein identification and quantification was performed using liquid chromatography tandem-mass spectrometry (LC–MS/MS). The data were analysed with MaxQuant v1.6.2.3 software. Bulk RNA sequencing (RNA-seq) was conducted using the Illumina HiSeq platform. Differentially expressed genes were identified using DESeq2. Additionally, we analysed publicly available single-cell (sc) RNA sequencing datasets (GSE109816, GSE121893) [1] on adult hearts from HF patients (n=6) and UDs (n=14) using Seurat package (V.2.3.4). Specific gene knockdown was achieved by siRNA transfection of human foetal cardiac fibroblasts (HCFs, Sigma), untreated or stimulated with TGF-β for 48-72h. The profibrotic marker expression was assessed using RT-qPCR and Western Blot. Cell viability was measured using PrestoBlue HS reagent (Invitrogen), and ATP production was quantified with CellTiter-Glo assay (Promega) in untreated and TGF-β-stimulated HCFs 48h after transfection. Results The LC–MS/MS analysis revealed 14 differentially expressed proteins (absolute log2FC&gt;1, adj. p&lt;0.05) in the HF group compared to UDs. The most upregulated protein in HF fibroblasts was dysferlin (DYSF, log2FC=5.78, adj. p&lt;0.004), which is known to play a role in the sarcolemma repair of both skeletal muscle fibres and cardiomyocytes. Bulk RNA-seq analysis identified a total of 67 significantly differentially expressed genes (absolute log2FC&gt;1, adj. p&lt;0.05). The comparative analysis of bulk RNA-seq results and publicly available scRNA-seq datasets revealed two commonly upregulated genes in HF fibroblasts or their subclusters, encoding transcription factor FOXF1 (log2FC=3.51, adj.p&lt;0.05) and matrix remodelling-associated protein MXRA5 (log2FC=2.91, adj. p&lt;0.05). Further in vitro studies on HCFs (n=4) showed that TGF-β upregulated DYSF (p&lt;0.001) and MXRA5 (p&lt;0.01) but downregulated FOXF1 (p&lt;0.05). DYSF silencing in HCFs (n=4) upregulated MXRA5 after 48h of TGF-β stimulation (p&lt;0.05), downregulated ACTA2 (48h and 72h of TGF-β stimulation, p&lt;0.05), and upregulated FOSL2 protein levels in untreated HCFs and 72h after TGF-β stimulation (n=3, p&lt;0.05). MXRA5 knockdown (n=8) resulted in the upregulation of DYSF (p&lt;0.05), ACTA2 (p&lt;0.05) and COL1A1 (p&lt;0.001) in untreated HCFs, and also upregulated DYSF (p&lt;0.01) and COL1A1 (p&lt;0.05) after 48h of TGF-β stimulation. FOXF1 silencing in HCFs (n=8) followed by 48h of TGF-β stimulation downregulated MXRA5 (p&lt;0.01) and ACTA2 (p=0.06), and upregulated DYSF (p&lt;0.001) and COL1A1 (p=0.05). Candidate targets knockdown reduced cell viability in untreated (n=4, DYSF : p&lt;0.01, MXRA5 : p&lt;0.001, FOXF1 : p&lt;0.01) and TGF-β stimulated (n=4, DYSF : p&lt;0.05, MXRA5 : p&lt;0.05, FOXF1 : p&lt;0.01) HCFs. ATP levels were decreased in TGF-β-stimulated HCFs after DYSF silencing (n=6, p=0.05). Conclusion Based on transcriptomics, proteomics and in vitro analysis of human cardiac fibroblasts, we identified DYSF , MXRA5 and FOXF1 as candidate targets implicated in profibrotic phenotype development, including profibrotic transcription factor FOSL2 regulation. These newly proposed candidates may serve as potential therapeutic targets for the treatment of cardiac fibrosis. References [1]Wang, L. et al. Nat. Cell Biol. 2020. Disclosure of Interests Ievgeniia Kocherova: None declared, Elena Pachera: None declared, Daria Nurzynska: None declared, Franca Di Meglio: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, 4P Science, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Przemyslaw Blyszczuk: None declared, Gabriela Kania: None declared.